Horseradish peroxidase C

Summary Horseradish peroxidase C (HRP; ferric) reacts with H₂O₂ to form Compound I, with an equilibrium constant of about 10¹⁴ M^–1. Two-step reduction of Compound I to Compound II and further to the ferric enzyme occurs reversibly at E_o values of 0.90 and 0.93 V (pH 7.0), respectively. The pH dependence of E_o values for each one-electron step, ferrous ferric Compound II Compound I indicates the presence of redox-linked ionization at pK_a values of 7.3 in the ferrous state, 11.0 in the ferric and 8.6 in Compound II. Zinc-substituted HRP C is oxidized to its free-radical form at an E_o value of 0.74 (pH 6.0). Comparison of oxidized zinc HRP C with Compound I shows that Compound I contains a porphyrin -cation radical. The flash photolysis study on the NO-ferric HRP C complex clearly indicates that the iron is pentacoordinated in HRP C while it is hexacoordinated in metmyoglobin. From the kinetic analysis of the acid-alkaline conversion of HRP C, the second-order rate constants of the reactions with H⁺ and HO^– are estimated to be 1.5 × 10¹⁰ and 6.7 × 10⁴ M^–1s^–1, respectively. The latter rate constant greatly varies with the kind of hemoproteins. In the presence of HRP C and O₂, indole-3-acetate is oxidized to its hydroperoxide form, which reacts effectively with HRP C to form Compound I and further converts Compound I to a verdohemoprotein.Abbreviations HRP horseradish peroxidase (HRP without subgroup letter denotes a classical preparation consisting of HRP B and HRP C) - EPR electron paramagnetic resonance - NMR nuclear magnetic resonance